Patients and sample collection

This study was approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (Ethics No: 2021004K). To determine whether anti-cancer treatment could induce cancer cell senescence in vivo and evaluate the clinical prognostic significance of TIS, two cohorts of CRC patients at Zhongnan Hospital of Wuhan University from January 2017 to December 2022 were collected.

In cohort 1, we collected paired colonoscopy biopsy specimens and radical resection specimens from 22 patients with locally advanced rectal cancer (LARC). Inclusion criteria: (1) The age range was from 18 to 75 years old, and gender was not restricted; (2) Colonoscopy diagnosed as CRC and the colonoscopic biopsy tissue specimens were available; (3) Neoadjuvant therapy was performed before radical surgery; (4) The radical resection specimens were available. Exclusion criteria: (1) History of radiotherapy and chemotherapy; (2) History of other malignant diseases; (3) History of bowel surgery.

Cohort 2 enrolled 30 patients with metastatic CRC (mCRC). Inclusion criteria: (1) The age range was from 18 to 75 years old, and gender was not restricted; (2) Patients with mCRC received first-line regimen contained irinotecan before surgery or biopsy; (3) The surgical resection specimens or colonoscopy biopsy specimens before anti-cancer treatment were available; (4) The biopsy spacimens or surgically resected tumors after irinotecan-based chemotherapy were available. Exclusion criteria: (1) History of radiotherapy and chemotherapy; (2) History of other malignant diseases; (3) History of bowel surgery.

Cell culture and senescence induction

Human CRC cell lines HCT116 and RKO, and human embryonic kidney cell line 293T were purchased from Procell Life Science & Technology Company (Wuhan, China). Cells were cultured in DMEM (Gibco, Carlsbad, CA) supplemented with 10% FBS (Gibco, Carlsbad, CA) at 37 °C with 5% CO2. For chemotherapy-induced senescence, 1.5 µM irinotecan (CPT-11) in HCT116 cells and 2.5 µM CPT-11 in RKO cells were used. For ionizing radiation (IR)-induced senescence, HCT116 cells were irradiated at different doses using the X-Ray Biological Irradiator (Precision X-Ray, Madison, USA). Cells were cultured for 96 h to allow the development of the senescent phenotype. Untreated CRC cells were cultured for 48 h and served as control.

Reagents and plasmids

CPT-11 (MCE, Shanghai, China) and TPX (MCE, Shanghai, China) were dissolved in DMSO (Beyotime, Shanghai, China) at the concentration of 40 mM and 50 mM, respectively. The stock solutions of CPT-11 and TPX were stored at -80 °C. Before use, the stock solution of CPT-11 was diluted with DMEM to 1.5 µM (for HCT116 cells) or 2.5 µM (for RKO cells), and the stock solution of TPX was diluted with DMEM to 20 µM. Plasmids of GFP-SERPINE1, p65-FLAG and mock vectors were purchased from Sino Biological (Sino Biological, Beijing, China).

Senescence-associated β-galactosidase (SA-β-Gal) staining

The activity of SA-β-Gal was determined using SA-β-Gal staining kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. The cultured cells were gently washed twice with PBS. Cells were fixed with the fixative solution and incubated with the SA-β-Gal staining solution mix overnight at 37 °C in an incubator without CO2. The SA-β-Gal staining solution mix was composed of X-Gal, staining solution and staining supplement. Under the catalysis of β-Gal, the X-Gal was generated into dark blue products. After washing with PBS, five random fields were captured under a microscope (Olympus IX73, Tokyo, Japan) for the analysis of the percentage of SA-β-Gal-positive cells.

CCK-8 assay

Cell viability was detected using CCK-8 assay (Beyotime, Shanghai, China). In brief, 5.0 × 103 CRC cells per well were seeded into 96-well plates. At the indicated time points, 10 µl CCK-8 solution was added into each well and incubated at 37 ℃ for 1 h in the dark. The absorbance was measured at a wavelength of 450 nm by a microplate reader.

EdU incorporation assay

BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, Shanghai, China) was utilized and EdU incorporation assay was performed according to the manufacturer’s protocol. The nuclei were stained with DAPI for 10 min in the dark and images were captured under a fluorescent microscope (Olympus IX73, Tokyo, Japan).

Colony formation assay

CRC cells were plated in 6-well plates at a density of 2 × 105 cells per well and treated with or without CPT-11 at the indicated concentrations for 96 h. Then, cells were trypsinized and re-seeded into 6-well plates (1.5 × 103 cells per well) and cultured for two weeks. After washing with PBS, cells were fixed with 4% paraformaldehyde (Beyotime, Shanghai, China) for 30 min at room temperature and stained with 0.1% crystal violet (Beyotime, Shanghai, China) for 15 min. The number of cell colonies was counted using Image J software (National Institutes of Health, Bethesda, MD, USA).

Wound-healing assay

To detect the impact of the EVs on the migration ability of CRC cells, the wound-healing assay was conducted as previously described [20]. Briefly, CRC cells were plated in 6-well plates. When the cells reached 80% confluence, a wound was created by manually scraping the cell monolayer with a 10 µl pipette tip. The cells were washed once with PBS to remove the debris and the medium was replaced with fresh medium (containing 0.5% FBS) supplemented with or without EVs (15 µg/ml). The area of migration was photographed at indicated time points and analyzed using Image J software.

Transwell assay

The transwell assay was conducted to evaluate the impact of EVs on the migration and invasion of CRC cells. For the migration assay, 5 × 104 CRC cells were seeded into the upper chambers (8 μm pore size, Corning, MA, USA) in serum-free DMEM. The lower chamber was filled with DMEM (2.5% FBS) with or without EVs (15 µg/ml). After incubation at 37 °C for 48 h, the migrated cells were fixed, stained, and photographed under a microscope (Olympus IX73, Tokyo, Japan). For the invasion assay, the upper compartment of the chamber was precoated with Matrigel (Corning, MA, USA) and the other steps were the same as the migration assay.

Flow cytometry

Cell cycle distribution was determined by flow cytometry as previously described [21]. In Brief, cells were incubated with DNA staining solution and permeabilization solution at room temperature for 30 min in the dark. Then, DNA content was measured by flow cytometry using a FACS Calibur system (Becton Dickinson, Franklin Lakes, NJ, USA), and the data were analyzed with FlowJo FACS analysis software (TreeStar, Ashland, OR, USA).

RT-qPCR

Total RNA was extracted from cells using Trizol reagent (Takara, Ohtsu, Japan) and reverse transcribed using the reverse transcription kit (Takara, Ohtsu, Japan). RT-qPCR was carried out with the SYBR Green Master Mix (Vazyme, Nanjing, China) on a CFX96 Touch System (Bio-Rad, CA, USA). β-actin was used as an internal control. Relative gene expression was calculated using the 2−ΔΔCT method. The primers were listed in Table S1.

Western blot

Protein was isolated from harvested cells or EVs by RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor Cocktail (MCE, Shanghai, China). The protein concentration was determined using the BCA protein assay kit (Beyotime, Shanghai, China) in accordance with the manufacturer’s instructions. Protein extracts were separated in 10% SDS-PAGE and transferred to PDVF membranes (Millipore, MA, USA). After blocking with 5% nonfat milk at room temperature for 2 h, the membranes were washed with TBST and incubated sequentially with primary antibody and secondary antibody. The protein signals were detected by enhanced chemiluminescence with ECL detection reagents on the chemiluminescence imager (Tanon, Shanghai, China). The primary antibodies used were listed in Table S2.

Immunofluorescent staining

Cells were fixed with 4% paraformaldehyde for 30 min and penetrated with Triton X-100 for 15 min. 10% goat serum (BOSTER, Wuhan, China) was utilized to block at 37 ℃ for 1 h. The cells were incubated sequentially with primary antibody at 4 °C for 16 h and secondary antibody at 37 ℃ for 1 h in the dark. Nuclei were counterstained with DAPI for 10 min. Images were captured by a confocal laser scanning microscope (SP8, Leica, Germany). The primary antibodies used were listed in Table S2.

Isolation of EVs

For chemotherapy-induced senescence, CRC cells were plated in 100 mm dishes and treated with CPT-11 (1.5 µM CPT-11 in HCT116 cells and 2.5 µM CPT-11 in RKO cells) for 96 h to induce senescence. To inhibit the expression of SERPINE1 in EVs from STCs, CRC cells were simultaneously treated with CPT-11 and TPX (20 µM) for 96 h. For IR-induced senescence, HCT116 cells were plated in 100 mm dishes and exposed to 10 Gy irradiation. Cells were cultured for 96 h to allow the development of the senescent phenotype. For control, HCT116 cells and RKO cells were plated in 10 mm dishes without treatment and cultured for 48 h to reach 90% confluence.

The cultured cells were washed twice with PBS to remove the drug and FBS and cultured in serum-free DMEM for 48 h, then the culture media was collected for EVs isolation and the number of cells in dishes was counted. The EVs were isolated by differential ultracentrifugation as described previously [22, 23]. Briefly, the harvested culture media was centrifuged at 300 g for 10 min followed by centrifugation at 2,000 g for 20 min to remove cellular debris and apoptotic bodies. The supernatant was transferred and centrifuged at 10,000 g for 30 min at 4 ℃ to isolate large EVs. Then the supernatant was filtered through a 0.22 μm polyethersulfone filter (Millipore, Merck Millipore, USA) to reduce potential large EVs contamination. The supernatant filtrate was subjected to high-speed ultracentrifugation at 100,000 g for 70 min at 4 ℃ with an SW28 rotor (Optima XE-100, Beckman Coulter, USA). Then the pellets were washed twice with PBS and ultracentrifuged at 100,000 g for 70 min at 4 ℃. The purified EVs were resuspended in sterile PBS and used for the following experiments.

Nanoparticle tracking analysis (NTA)

The concentration and size distribution of EVs were measured using a ZetaView nanoparticle tracking analyser (Particle Metrix, Meerbusch, Germany) equipped with ZetaView software (version 8.05.14 SP7). EVs were diluted to the appropriate concentration in PBS prior to measurement. The ZetaView system was calibrated using 110 nm polystyrene particles. NTA measurement was recorded and analyzed at 11 positions. The average concentration from three recordings was used as the EV concentration.

Transmission Electron microscopy (TEM)

The EVs samples were diluted in PBS before measurement. The EVs samples were added dropwise on 200 square mesh TEM grids and incubated for 10 min at 37 ℃. The grids were negatively stained with 2% phosphotungstic acid for 3 min, and imaged on a JEM1400 transmission electron microscope.

Proteinase K assay

EVs isolated from senescent HCT116 cells were resuspended in PBS and split into three identical aliquots. The mix was incubated at 37 ℃ with 0.5 mg/ml proteinase K in the presence or absence of 1% Triton X-100 for 30 min. For control, one of the aliquots was incubated without proteinase K. The samples were then boiled for 10 min and analyzed by western blot.

PKH67-labeled EVs transfer assay

CRC cells (5.0 × 104) were plated into 24-well plate. EVs (10 µg) released from senescent CRC cells were labeled with a PKH67 green fluorescence labeling kit (Sigma-Aldrich) [24]. The excess dye was removed through washing with PBS. The EVs were collected again by ultracentrifugation and resuspended in PBS. CRC cells were incubated with labeled EVs. After 18 h, cells were washed twice with PBS and stained with Hoechst 33342 for 10 min in the dark. Then cells were photographed under a fluorescent microscope (Olympus BX53, Tokyo, Japan).

iTRAQ-labeling quantitative proteomics analysis

EVs samples were isolated from senescent HCT116 cells and non-senescent HCT116 cells. Lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris-HCl, pH 8.5) with 1 × Cocktail (with EDTA) was added into each EVs sample. The mixtures were reduced with 10 mM DTT (37℃ for 30 min) and alkylated with 55 mM iodoacetamide (45 min in the dark). The samples were precipitated with ice-cold acetone (1:5, v/v) at -20 ℃ for 2 h and centrifuged at 25,000 g for 15 min. The pellets were air-dried and resuspended in lysis buffer followed sonicating (frequency of 60 Hz, for 2 min). The samples were centrifuged again, and the supernatant was transferred to a new tube. Protein quantitation was performed using a Bradford Protein Assay Kit.

The iTRAQ-based proteomic analysis was conducted by Beijing Genomics Institute. Briefly, proteins (100 µg) of each sample were digested using trypsin (1:20 w/w, Promega, Madison, USA) at 37 ℃ for 4 h. The digested protein peptide was desalted and labeled with iTRAQ reagents according to the kit protocol (Applied Biosystems, Foster City, USA). Then the peptides were pooled and purified using a 5 μm 4.6 × 250 mm Gemini C18 column with an LC-20AB liquid phase system (Shimadzu, Tokyo, Japan). The dried peptide samples were reconstituted with buffer A (5% ACN pH 9.8) and injected, eluting at a flow rate of 1mL/min by following gradients: 5% buffer B (95% ACN, pH 9.8) for 10 min, 5–35% for 40 min, 35–95% for 1 min, buffer B for 3 min, and 5% buffer B for 10 min. Finally, the eluted peptides were pooled into 20 fractions, which were then freeze-dried. The dried peptide samples were reconstituted with buffer A (2% ACN, 0.1% FA) for LC-MS/MS analysis. Separation was performed by Thermo UltiMate 3000 UHPLC. Peptides were separated with a gradient from buffer B (98% ACN, 0.1% FA) and the liquid gradient setting: 0–5 min, 5% buffer B; 5–45 min, 5–25% buffer B; 45–50 min, 25–35% buffer B; 50–52 min, 35–80% buffer B; 52–54 min, 80% buffer B; 54–60 min, 5% buffer B. Ultimately, the separated peptides were analyzed in Q Exactive HF-X (Thermo Fisher Scientific, San Jose, CA) for DDA (Data Dependent Acquisition) mode detection. Spectra generated by the Orbitrap was optimized by automatic gain control (AGC). The AGC was set to: MS1 3E6, MS2 1E5. The main parameters were set: ion source voltage was set to 1.9 kV, MS1 scanning range was 350–1,500 m/z; resolution was set to 60,000; MS2 starting m/z was fixed at 100; resolution was 15,000.

The raw MS/MS data were converted into MGF format by thermo scientific tool Proteome Discoverer, and the exported MGF files were searched using Mascot version 2.3.02 (Matrix Science, London, UK) against human Uniprot database (http://www.uniprot.org). The mass tolerance for precursor ions was set as 10 ppm and the mass tolerance for fragment ions was set as 0.02 Da. All identified proteins were required to have at least 2 peptides with at least one unique peptide. For quantitatively analyzing the peptides labeled with iTRAQ tags, the IQuant software was utilized. Proteins with fold change > 1.5 and Q value < 0.05 were considered differentially expressed.

Immunoprecipitation (IP) and mass spectrometer (MS) identification

Cells were lysed in IP lysis buffer, followed by incubation on ice for 15 min. Then the cell lysates were centrifuged at 12,000 g for 10 min. The supernatant was collected and incubated at 4 ℃ with primary antibody by a roller shaker overnight. Then 40 µl Protein A/G magnetic beads (MCE, Shanghai, China) were incubated with the cell lysates for 2 h at 4 °C. Beads were washed with PBST (PBS with 0.5% Triton X-100) three times. IP proteins were analyzed by western blot or LC-MS/MS analysis. The primary antibodies used were listed in Table S2.

Tumor xenograft experiments

The animal experiments were performed according to the approved study protocols by the Animal Ethics Committee of Wuhan University (approval number: ZN2023019). 5-week-old male BALB/c nude mice were housed and fed in specific pathogen free conditions. The mice were randomly assigned to four groups: PBS, Ctrl-EVs, Sen-EVs, and TPX-Sen-EVs. HCT116 cells (1 × 106 cells) together with or without EVs (50 µg for each mouse) were injected subcutaneously under the right armpits of the nude mice. The mice were intratumorally injected with EVs (50 µg for each mouse) every other day when the tumor volume reached 50 mm3. Mice injected with an equivalent volume of PBS following the same procedure served as control. Tumor volume calculations were obtained every 2 days using the following formula: Tumor volume (mm3) = (width2 × length)/2. To minimize animal suffering, once the tumor size of each group reached a significant difference, the mice were euthanized and tumors were excised and weighed. The xenograft tissues were fixed with 4% paraformaldehyde and embedded in paraffin for hematoxylin-eosin (H&E) and immunohistochemistry (IHC) staining.

H&E and IHC staining

For H&E staining, tumor tissues were fixed in formalin, embedded in paraffin, and cut into 5-µm-thick sections followed by H&E standard staining as described previously [21]. Images were taken under a microscope (Olympus BX53, Tokyo, Japan).

IHC staining was carried out as described previously [21]. Briefly, antigen retrieval was conducted by boiling the tissue sections with citrate buffer. Next, tissue sections were blocked in 10% goat serum for 1 h and incubated sequentially with primary antibody at 4 °C overnight and secondary antibody at 37 ℃ for 30 min. Images were captured on a microscope. The expression levels of p16, p21 and SERPINE1 were evaluated according to the immunoreactive score (IRS). The IRS was determined by the multiplication of staining distribution (0, less than 5%; 1, 5–25%; 2, 26–50%; 3, 51–75%; 4, more than 76%) and intensity score (0, no coloration; 1, pale yellow; 2, yellow; and 3, dark brown). IRS > 2 was considered positive. The primary antibodies used were listed in Table S2.

Statistical analysis

All statistical analyses were carried out with GraphPad Prism 8.0 (GraphPad Software, CA, USA). Categorical data were presented as numbers and proportions and analyzed with chi-square test. Continuous variables were expressed as mean ± standard deviation from at least three independent experiments. The comparison between two groups was conducted using Student’s t-test. One-way ANOVA with Bonferroni correction was used for multiple comparisons. Kaplan-Meier curve and log-rank test were used to calculate survival profiles. p value < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, and ***p < 0.001.